Metastasis is the major cause of therapeutic failure in cancer patients. A common site of metastases in humans is the lungs [19
]. In general, lung metastasis signifies widespread cancer with a poor survival rate, and living more than 5 years with metastatic cancer to the lungs is rare [20
]. Clearly, there is a need for therapeutics that combat metastatic cancer to the lungs and have low toxicity in normal tissues. One area of anticancer drug development that has received considerable attention over the past 2 decades is focused on targeting tumor angiogenesis and the unique features of tumor vasculature [5
]. Cancer cell survival, growth, and opportunity for metastasis depend on a tumor’s ability to attract and maintain a functional blood supply [21
]. In primary tumors, damaging the endothelium may impact a large number of cancer cells. Based on the observation that oxygen can diffuse a distance of ~100 to 150 μm, it is estimated that 10,000 malignant cells may be sustained for every millimeter of capillary growth in tumor angiogenesis [21
]. Furthermore, the irregularities in tumor microenvironments caused by aberrant vasculature may render tumor cells more likely to metastasize [22
]. Several reports have linked poor primary tumor oxygenation to development of metastases in soft tissue sarcoma, cervical carcinoma and squamous cell carcinoma of the head and neck [23
]. When applied in the clinic, small but encouraging clinical benefits have been observed when blood vessel directed therapies are combined with chemotherapy and radiotherapy leading to the inclusion of such agents in first or second line therapy for some malignancies [26
The focus of the present study was to investigate the growth and vascular development of squamous carcinoma cells seeding in the lungs. An experimental metastasis assay involving the injection of tumor cells directly into the bloodstream via the lateral tail vein was used. Though not including the entire metastatic cascade, the experimental lung metastasis model has several advantages. Because cells are directly injected into the bloodstream, control of the primary tumor is not necessary to evaluate the growth of metastases. By injecting a constant number of tumor cells, a predictable number of metastases are formed per animal. Even so when utilizing H&E stained sections to calculate lung tumor nodule volumes, from radii of sections separated by a known distance (), it became readily apparent ( and ) that there exists considerable variability in the time of establishment and subsequent growth of these metastases.
Moreover not all tumor nodules were spherical in shape, raising the question of whether non-spherical tumor nodules grew at a different rate than did spherical ones. However, statistical analysis of these two subsets revealed this not to be the case. The pooled results of all measurements () show how the median volume of SCCVII lung tumor nodules increased from 0.006 mm3 on day 7 to 0.51 mm3 on day 18.
Sections adjacent to those used for nodule measurement ( and ) were reserved for immunohistochemical analysis using the endothelial cell marker CD31. This methodology allowed the direct association of tumor nodule size and vascular development irrespective of the evaluation time point; an important consideration given that nodule size varied considerably at a particular assessment time point (). Vascular density determined using the previously described Chalkley counting technique [16
] showed that blood vessels were present in all tumor nodules even at the smallest detectable size (). However, closer examination of the data showed a consistent density of blood vessels in all SCCVII tumor nodules of sizes ≤ 0.5 mm3
, while the vascular density found in nodules greater than 0.5 mm3
increased with increasing nodule size. Again, as was the case for tumor growth (), when the effects of tumor nodule shape were considered, no difference in vessel density was seen between nodules classified as spherical or non-spherical ().
The present investigation utilized immunohistochemical and H&E staining to simultaneously assess the vascular development of multiple SCCVII lung metastases at various stages of growth. Irrespective of the day of assessment, tumor deposits ranging from the smallest size detectable to ≤ 0.5 mm3
always demonstrated similar blood vessel densities. This finding suggest that following blood borne dissemination, expanding SCCVII tumor cell populations are initially able to meet their nutritional and waste product removal needs by relying strictly on the preexisting lung vasculature, and tumor initiated blood vessel formation is not required. This is not however the case in larger metastases where increasingly greater blood vessel densities reflect an association between progressive tumor growth and additional vascular development. The induction of tumor vasculature first occurs in SCCVII lung metastases at a size of 0.5 to 1 mm3
and is independent of tumor nodule shape. The observation that blood vessel induction begins to take place in tumor deposits < 1 mm3
is consistent with prior window chamber tumor studies which reported that angiogenesis can be initiated by tumor cell numbers smaller than anticipated [29
In light of the importance of the vasculature to tumor cell survival and progression extensive efforts have been made to develop blood vessel directed therapies as anticancer treatments. Two fundamental treatment strategies have emerged [31
]; those interfering with tumor associated initiation of new blood vessels (antiangiogenic agents), and those targeting the established tumor blood vessel network (vascular disrupting agents). These two classes differ in three key respects: their physiologic target, the type or extent of disease that is likely to be susceptible, and the treatment scheduling [14
]. Indeed preclinical investigations have already established early stage tumors to be more susceptible to angiogenesis inhibition and advanced disease to be more efficiently targeted by vascular disrupting agents [32
]. Since metastases can simultaneously exist in organs at various stages of development (dormant, early phases of growth, well-established macroscopic neoplastic lesions), they will not only present with a variety of vascular developments but as a consequence will also be susceptible to different blood vessel directed therapeutics. The present methodology offers the opportunity to simultaneously assess the response of multiple metastases at various stages of growth to treatments targeting the tumor blood vessel network. Such information would provide valuable insights into treatment regimens which specifically target the tumor vasculature and aid the determination of which therapeutic interventions used alone or in combination might have particular utility in a metastasis setting.